Influenza drugs are few in number, and novel strains of the virus are resistant to many existing drugs. Thus, new drug targets are desperately needed. Influenza virus depends on cell membrane organization for entry through hemagglutinin-mediated fusion, and for assembly of viral components such as hemagglutinin (HA) and neuraminidase (NA) before budding. Cell membrane organization depends on cholesterol-rich membrane domains called "rafts," with which HA is associated, but the structure of these domains has been difficult to image directly because of the limits of light and electron microscopy for live cell imaging at nanometer resolution. Preliminary results show that HA clustering depends both on cholesterol and cortical actin, both of which are known to mediate cell membrane organization. We hypothesize that HA and NA clustering depend on cholesterol and cortical actin, and we will test this hypothesis using a novel super- resolution microscopy technique called fluorescence photoactivation localization microscopy (FPALM), developed in the Hess lab in 2006. FPALM will allow us to image individual molecules of HA or NA and actin cytoskeletal components, including actin- binding adaptor proteins, in living cells at physiological temperature, with resolution in the 10-40 nanometer range. Along with actin and tubulin, the candidate adaptor proteins were found in purified influenza virus as observed by mass spectroscopy. We will also test the hypothesis that HA and NA membrane clustering is mediated by interactions with one or more of these adaptor proteins. Finally, since the membrane organization of "rafts" is dependent on cholesterol and the cytoskeleton, we will test the hypothesis that drugs which reduce cell cholesterol and disrupt the actin cytoskeleton will affect HA and NA membrane clustering. The observation of a significant response to such drugs would constitute a major opportunity for development of a new class of drugs against influenza. PUBLIC HEALTH RELEVANCE: Cell membrane cholesterol and the cytoskeleton affect influenza membrane protein clustering needed for infection, so drugs which alter cholesterol and the cytoskeleton are good candidates as anti-viral therapies. We will use super-resolution fluorescence microscopy to test for interactions between viral components and the cytoskeleton, and to test the effects of these drugs on the assembly of viral components into membrane clusters. Drugs identified to have effects on viral protein clustering could be used as anti- viral therapies to reduce the spread of new strains of influenza and other viruses.